U.S. patent number 7,252,996 [Application Number 10/466,186] was granted by the patent office on 2007-08-07 for ancillary composition for the preparation of committed mature dentritic cells.
This patent grant is currently assigned to I.D.M. Immuno-Designed Molecules. Invention is credited to Jean-Pierre Abastado, Claire Boccaccio, Alessandra Nardin.
United States Patent |
7,252,996 |
Boccaccio , et al. |
August 7, 2007 |
Ancillary composition for the preparation of committed mature
dentritic cells
Abstract
The invention consists in the use of a maturation agent
comprising a mixture of ribosomal and/or membrane fractions for the
preparation of mature dendritic cells from immature dendritic
cells.
Inventors: |
Boccaccio; Claire (Paris,
FR), Nardin; Alessandra (Paris, FR),
Abastado; Jean-Pierre (Paris, FR) |
Assignee: |
I.D.M. Immuno-Designed
Molecules (Paris, FR)
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Family
ID: |
8182599 |
Appl.
No.: |
10/466,186 |
Filed: |
December 29, 2001 |
PCT
Filed: |
December 29, 2001 |
PCT No.: |
PCT/EP01/15314 |
371(c)(1),(2),(4) Date: |
December 19, 2003 |
PCT
Pub. No.: |
WO02/055675 |
PCT
Pub. Date: |
July 18, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040197901 A1 |
Oct 7, 2004 |
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Foreign Application Priority Data
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Jan 15, 2001 [EP] |
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01400109 |
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Current U.S.
Class: |
435/377; 435/372;
435/355 |
Current CPC
Class: |
C12N
5/0639 (20130101); A61P 31/00 (20180101); A61P
35/00 (20180101); A61K 38/217 (20130101); A61K
38/217 (20130101); A61K 35/74 (20130101); A61K
38/217 (20130101); A61K 2300/00 (20130101); C12N
2501/22 (20130101); C12N 2500/72 (20130101); C12N
2501/23 (20130101); C12N 2501/24 (20130101); A61K
2039/55511 (20130101); A61K 2039/5154 (20130101); C12N
2501/056 (20130101) |
Current International
Class: |
C12N
5/02 (20060101) |
Field of
Search: |
;435/375 ;424/93.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 99 47646 |
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Sep 1999 |
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WO |
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WO 02/074939 |
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Sep 2002 |
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WO |
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Other References
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Pascale Jeannin et al., "OmpA Targets Dendritic Cells, Induces
Their Maturation and Delivers Antigen into the MHC Class I
Presentation Pathway," Nature Immunology, V. 1, 2000, pp. 502-509.
cited by other .
Haeuw, Jean-Francois et al., "The recombinant Klebsiella
pneumoniaeouter membrane protein OmpA has carrier properties for
conjugated antigenic peptides", Eur. J. Biochem. 255, 1998, pp.
446-454. cited by other .
Nguyen, Thien Ngoc et al., "Chromosomal sequencing using a
PCR-based biotin-capture method allowed isolation of the complete
gene for the outer membrane protein A of Klebsiella pneumoniae",
Gene 210, 1998, pp. 93-101. cited by other .
Jongmans, Wim et al., "Th1-Polarizing Capacity of Clinical-Grade
Dendritic Cells is Triggered by Ribomunyl but Is Compromised by
PGE.sub.2 ", J Immunother, vol. 28, No. 5, Sep./Oct. 2005, pp.
480-487. cited by other .
Peng, Judy C. et al., "Generation and Maturation of Dendritic Cells
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other .
Barrou, Benoit et al., "Vaccination of prostatectomized prostate
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therapeutic use: Characterization and in vitro functional
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DC-based melanoma therapeutic vaccine", Sep. 5-7, 2005, Lisbon,
Portugal. cited by other .
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the invention: Antibody Dependent Cell Cytotoxicity (ADCC) assay",
APC USA 09/194,053, priority EP 96 401 099.5, May 21, 1996. cited
by other .
Coronel, Agnes et al., "Cytokine production and T-cell activation
by macrophage-dendritic cells generated for therapeutic use",
British Journal of Haematology, 2001, 114, pp. 671-680. cited by
other .
Tsuji et al::, "Maturation of Human Dendritic Cells by Cell Wall
Skeleton of Mycobacterium Bovis Bacillus Calmette-Guerin:
Involvement of Toll-Like Receptors", Infection and Immunity, vol.
68, No. 12, - Dec. 2000, pp. 6883-6890, XP000995248, ***See
abstract Materials and Methods, esp. p. 6884, RH column third
paragraph, Results, Discussion*** the whole document. cited by
other .
Banchereau and Steinman: "Dentritic Cells and the Control of
Immunity" Nature, vol. 392, Mar. 19, 1992, pp. 245-252, XP002134557
***see whole document, especially ***p. 246, column 2, paragraph 2.
cited by other .
Revillard et al.:, "In Vitro Cell Activating Properties of the
Composite Ribosomal Vaccine D53" Mem. Inst. Oswaldo Cruz, vol. 82,
No. Suppl. II, 1987, pp. 173-178, XP000995084, Rio de Janeiro ***
see whole document, especially *** p. 177, column 2, paragraph 1.
cited by other .
Caux et al.: "Gamma-Interferon Enhances Factor-Dependent Myeloid
Proliferation of Human CD34+ Hematopoietic Progenitor Cells" Blood,
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2630, column 2, paragraph 1. cited by other.
|
Primary Examiner: Ketter; James
Attorney, Agent or Firm: Young & Thompson
Claims
The invention claimed is:
1. A method for the preparation of mature dendritic cells from
immature dendritic cells, comprising treating said immature
dendritic cells with a maturation agent comprising a bacterial
mixture of ribosomal fractions or ribosomal and membrane fractions
to obtain mature dendritic cells.
2. The method according to claim 1, wherein the maturation agent
comprises interferon-.gamma. and a bacterial mixture of ribosomal
fractions or ribosomal and membrane fractions.
3. A process for the preparation of mature dendritic cells from
immature dendritic cells, said process comprising the step of
contacting in a culture medium immature dendritic cells with a
maturation agent comprising a bacterial mixture of ribosomal
fractions or ribosomal and membrane fractions to obtain mature
dendritic cells.
4. The process for the preparation of mature dendritic cells
according to claim 3, wherein the maturation agent comprises
interferon-.gamma. and a bacterial mixture of ribosomal fractions
or ribosomal and membrane fractions.
5. The process for the preparation of mature dendritic cells
according to claim 3, wherein ribosomal fractions or ribosomal and
membrane fractions are used at a dose of about 0.01 to about 100
.mu.g/ml.
6. The process according to claim 5, wherein ribosomal fractions or
ribosomal and membrane fractions are used at a dose of about 0.1 to
about 10 .mu.g/ml within the maturation medium.
7. The process according to claim 5, wherein ribosomal fractions or
ribosomal and membrane fractions are used at a dose of about 10
.mu.g/ml within the maturation medium.
8. The process for the preparation of mature dendritic cells
according to claim 5, said maturation agent comprising
interferon-.gamma. is at a dose of about 10 to 10000 UI/ml within
the maturation medium.
9. The process for the preparation of mature dendritic cells
according to claim 8, said maturation agent comprising interferony
is at a dose of about 100 to about 1000 UI/ml within the maturation
medium.
10. The process for the preparation of mature dendritic cells
according to claim 8, said maturation agent comprising
interferon-.gamma. is at a dose of about 1000 UI/ml within the
maturation medium.
11. A method for the preparation of mature dendritic cells from
immature dendritic cells, comprising treating said immature
dendritic cells with a maturation agent comprising a bacterial
mixture of ribosomal fractions or ribosomal and membrane fractions,
and wherein the ribosomal fractions are from bacterial strains
selected from the group consisting of Klebsiella pneumoniae,
Streptococcus pneumoniae, Streptococcus pyogenes group A and
Haemophilus influenzae to obtain mature dendritic cells.
12. The method according to claim 11, wherein the maturation agent
comprises interferon-.gamma. and a bacterial mixture of ribosomal
fractions or ribosomal and membrane fractions.
13. The process for the preparation of mature dendritic cells
according to claim 11, wherein the maturation agent comprises
interferon-.gamma. and a bacterial mixture of ribosomal fractions
or ribosomal and membrane fractions.
14. The process for the preparation of mature dendritic cells
according to claim 11, wherein the membrane fractions are from
Klebsiella pneumoniae.
15. The process for the preparation of mature dendritic cells
according to claim 11, wherein ribosomal fractions or ribosomal and
membrane fractions are used at a dose of about 0.01 to about 100
.mu.g/ml.
16. The process according to claim 15, wherein ribosomal fractions
or ribosomal and membrane fractions are used at a dose of about 0.1
to about 10 .mu.g/ml within the maturation medium.
17. The process according to claim 15, wherein ribosomal fractions
or ribosomal and membrane fractions are used at a dose of about 10
.mu.g/ml within the maturation medium.
18. The process for the preparation of mature dendritic cells
according to claim 11, said maturation agent comprising
interferon-.gamma. is at a dose of about 10 to 10000 UI/ml within
the maturation medium.
19. The process for the preparation of mature dendritic cells
according to claim 18, said maturation agent comprising
interferon-.gamma. is at a dose of about 100 to about 1000 UI/ml
within the maturation medium.
20. The process for the preparation of mature dendritic cells
according to claim 18, said maturation agent comprising
interferon-.gamma. is at a dose of about 1000 UI/ml within the
maturation medium.
Description
The present invention relates to a method for the production of
committed mature dendritic cells (DCs) and particularly to a
cocktail of ribosomal and/or membrane bacterial extracts used for
the DCs maturation.
Dendritic cells are defined as the most potent antigen presenting
cells able to stimulate both primary and secondary immune responses
against specific exogenous antigen (Hart "Dendritic cells: unique
leucocyte populations which control the primary immune response"
Blood, 1997, vol. 90, p3245). In vivo, immature dendritic cells
that have captured antigens in the periphery migrate through
lymphatic vessels to T cell zones of lymphoid organs where they
present epitopes deriving from these antigens in the context of MHC
molecules and allow activation and proliferation of
antigen-specific naive T cells.
Stimulated lymphocytes can be cytotoxic or auxiliary, but also
regulatory or suppressive lymphocytes, depending on the type of the
dendritic cells and on the pre-existing cytokine pattern.
During migration, dendritic cells undergo a maturation process that
results in morphological and phenotypical changes. Maturation
induces a reduced capacity of DCs to capture antigens and an
increased capacity of antigen presentation. Maturing DCs express
higher levels of costimulatory molecules, acquire the expression of
CD83 on their surface, they produce cytokines stimulating effector
T cell subtypes and acquire migratory abilities (for a review, see
"Immunobiology of dendritic cells", Banchereau et al., 2000, Ann.
Rev. Immunol., 18:767-811).
Possibilities for preparing ex vivo large quantities of dendritic
cells have recently been developed, followed by a growing interest
for the use of these cells in immunotherapy and as cellular
vaccines.
Dendritic cells can be obtained from different tissue sources or
form precursors present in blood or in bone marrow. Immature
dendritic cells may be obtained from blood cells by differentiating
monocytes using defined culture conditions (Boyer et al.,
"Generation of phagocytic MAK and MAC-DC for therapeutic use:
Characterization and in vitro functional properties", Exp. Hematol.
1999, vol.27, pp751-761). Proliferating dendritic cells progenitors
have also been identified within the small CD34+ subfraction of
cells in human blood (Inaba et al. "Identification of proliferating
dendritic cells precursors in mouse blood", 1992, J. Exp. Med.,
vol.175, p1157) and methods have been developed to differentiate
these cells.
After being differentiated from blood monocytes, for example in
presence of IL-13, dendritic cells present an immature phenotype:
they are powerful for the antigen capture by pinocytosis or
phagocytosis, exhibit low levels of the costimulation molecule CD80
and do not express the surface marker CD83 Moyer et al, 1999).
Mature dendritic cells are more potent immune modulators than
immature DCs. In particular, the capacity of dendritic cells to
induce an immune response in vivo has been correlated to their
degree of maturation (Labeur M. S et al "Generation of tumour
immunity by bone-marrow derived dendritic cells correlates with
dendritic cell maturation stage", J. Immunol., 1999,162,
168-175).
There are several known agents used for the maturation of DCs for
research purposes, such as poly IC, ligands of CD40, anti-CD40
antibodies, endotoxins, living bacterias, culture supernatants and
cocktail of agonistic cytokines, including TNF.alpha.. However,
clinical trials for which patients are vaccinated with mature
dendritic cells are under development, using cells presenting
foreign antigens on their surface after being pulsed with peptides
or loaded with particulate antigens. It is therefore required to
develop reproducible clinical grade maturation conditions to obtain
of committed mature dendritic cells with defined immunomodulatory
capacity.
Mycobacterium bovis bacillus Calmette-Guerin (BCG) is shown to
activate dendritic cells (Kim et al., "Enhanced antigen-presenting
activity and tumour necrosis factor a-independent activation of
dendritic cells following treatment with Mycobacterium bovis
bacillus Calmette-Guerin", Immunology, 1999, vol.97, pp 626-633).
However, BCG is a living attenuated bacterial strain, and its use
in a cellular vaccine presents several drawbacks including safety
concerns.
Ribomunyl.RTM. (International Non-proprietory Name, or Generic
name: Ribosomal and membranar bacterial fractions, membranar
proteoglycanes) is known for its non specific natural
immunostimulatory effect. It contains both proteoglycans from
Klebsiella pneumoniae (0.015 mg in a dose of lyophylisate) and
ribosomal fractions containing 70% RNA from 4 different bacterial
strains, Klebsiella pneumoniae (35 parts), Streptococcus pneumoniae
(30 parts), Streptococcus pyogenes group A (30 parts) and
Haemophilus influenzae (5 parts) (0.01 mg of ribosomal extracts in
a dose of lyophylisate). The proteoglycans act as an adjuvant and a
nonspecific immunostimulant, whereas the immunogenicity of the
ribosomes is attributed either to peptides naturally bound to
ribosomes or to epitopes bound to membrane and cytoplasmic
ribosomes (Clot et al "Pharmacology of ribosomal immunotherapy",
Drugs, 1997, vol.54, suppl.1, pp 33-36). Ribomunyl.RTM., also
designed as RBL, triggers mucosal immune responses (Bene &
Faure, "From Peyer's patches to tonsils", Drugs, 1997, 54, suppl.1,
pp24-28). RBL was shown to stimulate the general innate immune
response by acting on polymorphonuclear cells (PMNs) and
macrophages, to increase the production of several cytokines (IL-1,
IL-6, IL-8, TNF.alpha., CSF), and to be able to activate natural
killer cells.
The aim of the present invention is to provide a new process for
the preparation of mature dendritic cells from immature dendritic
cells. This process comprising the step of contacting, in a culture
medium, immature dendritic cells with a maturation agent comprising
a bacterial mixture of ribosomal and/or membrane fractions.
The term "maturation" is defined as the action on immature highly
phagocytic dendritic cells, resulting into phenotypic and/or
functional modification of the cells. The associated phenotypic
modifications are the increase in CD86, CD86, CD83, MHC class I and
II molecules cell surface expression and the decrease in CD14
surface expression. The functional changes may be the loss of
phagocytic properties, the acquisition of migration abilities, an
increased allogeneic T cells stimulation efficiency and changes in
the cytokine and chemokine expression profile, and particularly an
increased IL-12 secretion. The IL-12 production by DCs is critical
for their in video function, since this cytokine has been shown to
generate a polarization of the immune response towards the Th1
pathway in vivo. A Th1 type response is considered as immune
response, involving stimulation of antigen specific T lymphocytes
CD8+, whereas a Th2 type immune involves rather a stimulation of
antibody response and eventually unresponsiveness of the cytotoxic
lymphocytes to an antigen.
The term "committed DCs" is defined as mature DCs directing the
immune response clearly towards Th1 immunostimulation or towards
immunoregulation.
The term "ribosomal extracts" is defined as bacterial extracts
containing ribosomal fractions, and particularly single and/or
double stranded ribonucleic acid. Ribosomal extracts or fractions
correspond to any extract containing ribosomes, purified or
partially purified from a bacterial culture. The process of
preparation of such extracts comprises at least a step of lysis of
the bacteria obtained after the culture, and a step of separation
of the fraction containing bacterial ribosomes from the total
lysate, in particular by centrifugation or filtration.
The term "membrane extracts" is defined as bacterial extracts
enriched in membrane fractions. Membrane extracts or fractions
correspond to any extract or fraction containing membranes,
purified or partially purified from a bacterial culture. The
process of preparation of such extracts comprises at least a step
of lysis of the bacteria obtained after the culture, and a step of
separation of the fraction containing bacterial membranes, in
particular by centrifugation or filtration.
The different bacterial fractions can be prepared according to
methods known by a man skilled in the art, such as a method
described by Haeuw J. F. et al (Eur. J. Biochem., 255, 446-454,
1998), or such as a method described in U.S. Pat. No. 4,249,540,
filed by Pierre Fabre S. A.
The term "bacterial mixture" is defined as a mixture of bacterial
extracts, possibly originating from different bacterial strains,
comprising membrane and ribosomal fractions.
The term "maturation medium" is defined as a culture medium
appropriate for the cells survival and differentiation, in which is
added a maturation agent, such a culture medium being liable to be
supplemented.
According to an embodiment of the invention, the process for the
preparation of mature dendritic cells is characterized in that the
maturation agent comprises a bacterial mixture of ribosomal and
membrane fractions and interferon-.gamma. (IFN-.gamma.). The
interferon-.gamma. has a synergistic effect to the maturation agent
to increase maturation characteristics of DCs and their stimulating
phenotype. A "stimulating phenotype" of the dendritic cells is
defined as inducing Th1 response and secreting a cytokine profile
favouring cytotoxic T lymphocytes.
According to another embodiment of the invention, the process is
characterized in that immature dendritic cells are contacted with a
maturation agent comprising membrane fractions being from
Klebsiella pneumoniae. Such maturation agents may be used at
concentrations from about 0.01 to about 100 .mu.g/ml, and
preferably of about 0.1 to about 10 .mu.g/ml in the maturation
medium.
According to another embodiment of the invention, the process is
characterized in that immature dendritic cells are contacted with a
maturation agent comprising ribosomal fraction being from the
bacterial strains Klebsiella pneumoniae, Streptococcus pneumoniae,
Streptococcus pyogenes group A and Haemophilus influenzae. Such
maturation agents may be used at concentrations from about 0.01 to
about 100 .mu.g/ml, and preferably of about 0.1 to about 10
.mu.g/ml in the maturation medium.
In a particular embodiment of the invention, immature dendritic
cells are contacted with a maturation agent comprising a bacterial
mixture of ribosomal and membrane extracts used at a dose of about
0.01 to about 100 .mu.g/ml, and preferably of about 0.1 to about 10
.mu.g/ml in the maturation medium.
In a particular embodiment of the invention, the maturation agent
is Ribomunyl.RTM. (Inava Laboratory, Pierre Fabre).
In another particular embodiment of the invention, immature
dendritic cells are contacted with a maturation agent comprising a
bacterial cocktail of ribosomal and membrane extracts and
interferon-.gamma. at a dose of 10 to 10000 UI/ml, and preferably
from about 100 to about 1000 UI/ml. Ability of IFN-.gamma. to
increase the maturation effect was tested and found to be effective
in particular on the dendritic cells IL-12 secretion (see example
3). When comparing the effects on the cytokine secretion of
contacting immature dendritic cells with a maturation agent
comprising Ribomunyl.RTM. (RBL) alone, or with a maturation agent
comprising RBL and IFN-.gamma., or with a known maturation agent,
such as poly I:C combined to anti-CD40, the contact of cells with
RBL and IFN-.gamma. leads to an IL-12 secretion level superior to
that obtained with the other maturation agents, and the contact of
cells with RBL induces an IL-10 secretion level superior to the
other maturation agents (see example 3). The ratio between the
level of IL-12 secretion and the level of IL-10 secretion shows
that when both RBL and IFN-.gamma. are used, the cells are
effectively committed towards an immunostimulating mature dendritic
cells phenotype.
On the other hand, the obtention of DCs having a higher IL10
secretion level and a lower IL12 secretion level, using for example
RBL alone as a maturation agent, leads to mature dendritic cells
possessing interesting immunoregulatory properties, in particular
to control auto-immune diseases.
The present invention also relates to a process for the preparation
of antigen loaded mature dendritic cells. Cells may be antigen
loaded by phagocytosis, pinocytosis, affinity binding, fusion,
nucleic acid (DNA, RNA) transfer or receptor mediated uptake,
according to methods known by a man skilled in the art. The
dendritic cells culture medium may be completed with soluble or
particulate antigens, including tumour target cells or cell debris,
or specific peptides against which an immune response is expected.
The culture medium may also be supplemented with genetic material
coding for a peptide or a protein against which a modulation of the
immune response is desired, this genetic material being linked to a
vector able to allow the transfection of the dendritic cells.
Where it is desirable for cells to take up antigens by
phagocytosis, it is preferable to add antigen to the culture of
immature dendritic cells prior to addition of the dendritic cells
maturation factor. Phagocytosis may be desirable when particulate
antigens, cell lysates or immune complexes are used. When soluble
peptide antigens are used, it is preferable to expose the antigen
to dendritic cells after a meanwhile maturation.
The present invention also concerns the dendritic cells liable to
be obtained according to the process described in the present
application.
Immature DCs might be obtained by any method known by a man skilled
in the art. As an example monocyte derived dendritic cells can be
prepared according to patent applications WO 94/26875, WO 96/22781
or WO 97/44441. Dendritic cells may also been prepared according to
Banchereau et al ("Immunobiology of dendritic cells" Annu. Rev.
Immunol., 2000, 18:767-911).
Dendritic cells liable to be obtained according to the process of
the invention are able to act on precise T cells subpopulations.
This means that the dendritic cells according to the invention are
able to stimulate or to regulate Th2/Th1 immune response. DCs are
able to induce in vivo antigen-specific proliferation of T cells,
thus leading to antigen specific increased cytotoxicity and
immunostimulation, or to induce in vivo regulatory T cells and
therefore inhibition of antigen-specific cytotoxic T cells, leading
to unresponsiveness to a specific antigen. The balance between
immunostimulatory and immunoregulatory capacity of the mature
dendritic cells depends on the maturation conditions applied to the
immature cells and on the type of DCs submitted to these
conditions.
A induced immune response might be characterized by an in vivo
clinical immune response against a given pathogen or a tumour,
leading to its decrease or its elimination. In vitro, this may be
measured, for dendritic cells, in a immunostimulation assay of
antigen-specific cytotoxic T lymphocytes. The functionality of
dendritic cells treated according to the invention may be measured
as their target recognition capacity and by an analysis of their
cytokine and chemokine release. A regulated immune response might
be observed clinically, in the case of an auto-immune disease, by
the decrease or disappearance of the symptoms. In vitro, antigen
presenting cells able to regulate an immune response are
characterized by their reduced secretion of stimulatory cytokines
(IL-1, IL-12, IFN-.gamma.) and their increased secretion of certain
inhibiting cytokines (IL 10, TGF-.beta.).
The cell morphology of dendritic cells after treatment with
Ribomunyl.RTM. and IFN.gamma. is characterized by the presence of
dendrites, whereas they are not visible on immature dendritic cells
Banchereau et al., 2000).
The flow cytometry analysis of the phenotype of the cells after a
40 hours contact with a maturation agent comprising either RBL
alone or RBL plus interferon-.gamma. shows that the dendritic cells
population is only partially maturated, as evidenced for example by
the CD83 expression pattern (see FIG. 3). This partial maturation
could possibly allow the dendritic cells to pursue their maturation
process in vivo, after being injected to the patient. In
particular, such cells could be able to efficiently migrate from
their injection point to lymph nodes. This property may open
interesting possibilities for in vivo therapeutic applications.
The examples cited in the present application indicate a contact
between dendritic cells and the maturation agent during 40 hours.
However the process according to the invention may comprise a step
of contacting immature dendritic cells with a maturation agent
during 18 hours or even during a shorter time, according to the
expected maturation level of the dendritic cells.
Dendritic cells liable to be obtained according to the process are
usable for immunotherapy and for vaccinology. Administration of the
cells to a patient is possible, the cells and additives being of
clinical grade.
The present invention also concerns pharmaceutical compositions
containing as active substance dendritic cells liable to be
obtained according to the process described in the application.
The present invention also concerns cellular vaccine composition
containing as active substance dendritic cells liable to be
obtained according to the process described in the application.
Pharmaceutical compositions, cellular vaccine compositions and
immunotherapeutic drugs containing antigen presenting cells
prepared according to the methods described might be administered
to patients under various galenic forms comprising the intradermal,
subcutaneous, intraveinous, intralymphatic, intranodal,
intramucosal or intramuscular administration The number of
dendritic cells in a single dose injected is comprised from about
10.sup.6 to about 10.sup.9 cells for a patient, and preferably from
about 10.sup.7 to about 10.sup.8 cells for a patient for a single
dose. As an example, the patient may receive one injection each
week, during six successive weeks.
DESCRIPTION OF THE FIGURES
FIG. 1: Ribomunyl.RTM. dose response for dendritic cells
maturation.
Immature DCs were incubated during 40 hours in the presence of
doses ranging from 0.001 to 10 .mu.g/ml of Ribomunyl. To follow
maturation, DCs were stained with anti-CD83 antibodies, anti-CD86
and anti-HLA ABC antibodies and the fluorescence analysed with flow
cytometer.
The X axis represents the doses used of Ribomunyl, in .mu.g/ml
within the maturation medium. The expression of the markers is
expressed, for CD86 and HLA-ABC markers, as Mean Fluorescence
Intensity arbitrary units (left Y axis) and for CD83 marker as
percentage of cells expressing the markers (right Y axis). The
clear and dark histograms corresponds respectively to the CD86 and
to the HLA-ABC expression, whereas the curve represents the
percentage of cells expressing CD83 marker.
FIG. 2: DCs recovery and mortality upon different maturation
conditions.
Immature DCs were incubated for 40 hours in the presence of
standard reagents anti-CD40 (3 .mu.g/ml) and poly(I:C) (100
.mu.g/ml), in the presence of the clinical grade reagents Ribomunyl
(1 .mu.g/ml) or Ribomunyl (1 .mu.g/ml) and IFN-.gamma. at 1000
UI/ml. Cell recovery after culture was estimated by counting living
cells under the microscope on Malassez slide. Cell viability was
measured by FACS using TOPRO-3 technology (Molecular Probes)
The X axis represents the different maturation condition used,
whereas the Y axis represents the percentage of cells recovered or
the percentage of cells mortality. The histograms correspond to the
expression of cells recovery, the lozenges correspond to the
percentage of cell mortality.
FIG. 3: Phenotypic FACS analysis of DCs without any maturation
treatment and after incubation with different maturation
agents.
Immature DCs were incubated for 40 hours in the presence of
standard reagents anti-CD40 (3 .mu.g/ml) and poly(I:C) (100
.mu.g/ml), in the presence of the clinical grade reagent Ribomunyl
(1 .mu.g/ml) or Ribomunyl (1 .mu.g/ml) and IFN-.gamma. at 1000
UI/ml. The fulllines corresponds to an isotype control. The dotted
lines correspond to the samples analysis.
FIG. 4: T cells allogeneic stimulation of mature DCs
Allogeneic mixed lymphocyte reaction (MLR) was performed on DCs
matured either by treatment with clinical grade RBL (1 .mu.g/ml),
RBL (1 .mu.g/ml)+IFN-.gamma. (1000 U/ml), poly I:C+anti-CD40 or
with IFN-.gamma. alone (1000 UI/ml) and on immature DCs. DCs were
incubated during 5 days with a fixed number of allogeneic T
lymphocytes present in a peripheral blood leukocyte extract. Cell
proliferation was quantified by [.sup.3H] thymidine uptake of cells
incubated with 1 .mu.Ci of [methyl-3H] thymidine during the last 18
hours of culture.
The X axis represents the different DCs/T cells ratios, whereas the
Y axis corresponds to the quantification of [.sup.3H] thymidine
uptake of the cells. Dark lozenges represent values obtained with
non treated DCs (=immature DCs), white circles represents values
treated with IFN-.gamma., black triangles correspond to cells
treated with RBL alone, white triangles correspond to cells treated
with RBL+IFN-.gamma., and black squares correspond to cells treated
with poly I:C and anti CD40.
FIG. 5: IL12 secretion of mature DCs
Culture supernatants, after 40 hours of cells culture at 2.10.sup.6
DCs/ml, were assayed by commercial ELISA kits for IL-12 p70
cytokine secretion.
The X axis indicates the different maturation conditions used, as
well as the control (non treated cells), the Y axis represents the
quantity of IL12p70, in pg/ml in the culture supernatant.
FIG. 6: IL10 secretion of mature DCs
Culture supernatants, after 40 hours of cells culture at 2.10.sup.6
DCs/ml, were assayed by commercial ELISA kits for EL-10 cytokine
secretion.
The X axis indicates the different maturation conditions used, as
well as the control (non treated cells), the Y axis represents the
quantity of IL10, in pg/ml in the culture supernatant.
EXAMPLES
Example 1
Ribomunyl.RTM. Dose Response for Dendritic Cells Maturation
Dendritic Cells
Immature dendritic cells (DCs) were prepared by culture of
peripheral blood monocytes and elutriated, according to the patent
application WO 97/44441 and to Boyer et al. ("Generation of
phagocytic MAK and MAC-DC for therapeutic use: Characterization and
in vitro functional properties" Exp. Hematol., 1999, 27, 751-761).
DCs were differentiated in AIMV medium supplemented with 500 U/ml
GM-CSF (Leucomax, Novartis Pharma) and 50 ng/ml IL13 (Sanofi
Synthelabo) (=complete AIMV medium), and elutriated after 7 days of
culture. 2.10.sup.7 DCs/ml were then suspended in human albumin 4%
and 10% dimethylsulfoxide (DMSO) and frozen in liquid nitrogen.
Maturation
The day before maturation, DCs were thawed and left overnight in
complete AIMV medium containing 500 U/ml GM-CSF and 50 ng/ml IL13.
Ribomunyl (RBL) (Inava laboratory, Pierre Fabre) was purchased in a
pharmacy. Each vial of lyophilised RBL contains 0.010 mg of
ribosomal fractions and 0.015 mg of membrane fractions. RBL is
resuspended in AIMV medium (0.1 mg/ml of active fractions)
extemporarily. 2.10.sup.6 DCs/ml were then incubated for 40 hours
in the presence of doses of 0.001-0.01-0.1-1-and 10 .mu.g/ml of
Ribomunyl.
Cell Morphology
Morphology of dendritic cells obtained by blood monocytes
differentiation and further treatment with Ribomunyl plus
IFN-.gamma. shows the apparition of dendrites, which are
characteristics of mature DCs, those dendrites are not visible when
immature DCs are observed (data not shown).
Phenotypic Analysis
To follow maturation, DCs were analysed for their expression of
CD83, CD86 and HLA ABC markers. DCs were suspended in phosphate
buffer saline (PBS) supplemented with foetal calf serum serum 1%,
at 4.10.sup.6 cells/ml. 100 .mu.l of cell suspension (4.10.sup.5
cells in each tube) were incubated on ice in the dark for 30 min
with fluorochrome conjugated monoclonal antibody: 10 .mu.l of
PE-conjugated mAb anti-CD83, 10 .mu.l of PE-conjugated mAb
anti-CD86 or 10 .mu.l of FITC-conjugated anti-HLA ABC (Immunotech,
Marseille, France). Cells were then washed again in PBS,
centrifuged at 1400 rpm for 5 min at 20.degree. C. and resuspended
in PBS containing TO-PRO 3 at 3 nM, to exclude death cells from
analysis.
Flow cytometry analysis was performed with a Becton Dickinson
cytometer with a CellQuest software.
Results:
Results are presented on FIG. 1. Expression of CD86 and HLA-ABC
markers is indicated as Mean Fluorescence Intensity arbitrary units
(left Y axis) and expression of CD83 marker is indicated as a
percentage of cells expressing the marker (right Y axis).
A range between 1 ng/ml and 10 .mu.g/ml of Ribomunyl.RTM. is tested
to evaluate the optimal concentration of RBL able to induce DCs
maturation after 40 hours of culture with the lowest possible
toxicity.
Between 1 ng/ml and 0,1 .mu.g/ml, no maturation is obtained based
on CD14, CD83, HLA-ABC and CD86 expression. Above 1 .mu.g/ml,
apparition of CD83 expression, up-regulation of CD86 and HLA ABC
are seen. The cell mortality does not increase regardless of the
RBL concentration used (not shown). A RBL dose of 1 .mu.g/ml for 40
hours of maturation is therefore chosen for further studies.
Example 2
Analysis of DCs Prepared According Different Conditions Morphology
of the Cells, Recovery, Mortality and Capacity to Induce T Cell
Proliferation.
Cells
Immature dendritic cells were prepared as described in example
1.
2.10.sup.6 DCs were incubated in complete AIMV medium for 40 hours
in the presence of standard maturation reagents anti-CD40 (3
.mu.g/ml) and poly(I:C) (100 .mu.g/ml), in the presence of the
clinical grade reagent Ribomunyl.RTM. (RBL, 1 .mu.g/ml), or in the
presence of RBL(1 .mu.g/ml) and IFN.gamma. (Imukin, 1000 U/ml).
Culture was done in 24 well plates with 2.times.10.sup.6
DCs/ml.
Cell Recovery and Mortality
Cell recovery after culture was estimated by counting living cells
on Malassez slide. Viability of the cells is assessed by Trypan
blue exclusion. Cell viability was also measured by FACS using
TOPRO-3.
Phenotypic Analysis
DCs phenotype was determined by double staining flow cytometry
analysis using the same markers CD83, CD86 and HLA ABC and using
the same conditions than example 1, plus CD14 marker (10 .mu.l of
FITC-conjugated mAb anti-CD14, Becton Dickinson).
Allogeneic Mixed Lymphocyte Reaction (MLR)
Variable numbers of DCs (non treated, clinical grade reagent
treated, or poly I:C+anti-CD40 treated) were incubated for 5 days
with a fixed number of allogeneic T lymphocytes. Cell proliferation
was quantified by [.sup.3H] thymidine uptake of cells incubated
with 1 .mu.Ci of [methyl-3H] thymidine during the last 18 hours of
culture.
Results:
Cell Recovery and Mortality
The number of DCs harvested after RBL treatment during 40 hours is
1,5 fold lower than non treated DCs (FIG. 2). Cell mortality is
expressed as a percentage of recovered cells.
The cell recovery after the treatment with RBL or with
RBL+.gamma.IFN was inferior to that of non treated cells, the
addition of IFN.gamma. does not significantly modify the cell
recovery, which remains superior to cell recovery after poly I:C
plus anti CD40 treatment.
Phenotypic Analysis
DCs phenotype (FIG. 3) shows an increase of CD83 marker on the cell
surface after RBL and after RBL+IFN-.gamma. treatment. After RBL
treatment, the bimodal pattern of CD83 expression shows the
presence of two populations of cells, differing by their CD83
expression levels. The CD83 level of expression on RBL+IFN-.gamma.
treated cells is comparable to that of poly I:C+anti CD40 treated
cells, indicative of mature cells.
Allogeneic Mixed Lymphocyte Reaction (MLR)
FIG. 4 shows that in mixed lymphocyte reaction, RBL treated DCs
were 5 fold more potent stimulators than untreated or .gamma.IFN
alone treated DCs. This stimulation is similar to that obtained
with DCs treated with poly I:C plus anti CD40.
The addition of IFN-.gamma. to RBL did not significantly increase
the DCs capacity to induce allogeneic T cell proliferation.
Example 3
Cytokine Secretion of Dendritic Cells Matured upon Different
Conditions
Cells
Immature dendritic cells were prepared as described in example 1.
2.10.sup.6 DCs were matured in the same conditions as the previous
example.
Cytokine Detection
Culture supernatants, after 40 hours of cells culture at 2.10.sup.6
DCs/ml, were assayed by ELISA for IL-12 p70 and IL-10 cytokine
secretion by commercial ELISA.
Cytokine production was measured in the supernatants of DCs culture
using matched antibodies specific for IL12p70 (MAB611, BAF219) and
for IL10 MAB217, BAF217). The assays were performed according to
the manufacturer's instructions (R & D systems).
Results
RBL treatment induces production of small amounts of IL12p70.
Addition of IFN-.gamma. and RBL to DCs induced a 10 fold increase
in the IL12p70 cell secretion. This value is 4 fold higher than the
one obtained with poly I:C plus anti-CD40 treatment (FIG. 5).
RBL induced secretion of large amount of IL10. Addition of
IFN-.gamma. and RBL to DCs significantly decreases the level of
IL10 secreted by the cells, when compared to IL10 secretion by RBL
treated cells. High levels of IL12p70 are detected in supernatants
of RBL plus IFN-.gamma. treated DCs. The high level of IL10
produced by RBL treated DCs treatment significantly decreases upon
IFN-.gamma. addition (FIG. 6).
* * * * *